The objective of trapping and enumeration of air-borne fungal spores in indoor environments may be 1) to determine the load of allergenic spores and their composition, 2) to determine whether there were hidden sources of fungal amplification, 3) to assess the effectiveness of remediation. Regardless of the objective, the methods of trapping and enumeration are the same.

Fungal spores trapping

Generally fungal spores are trapped by impacting air onto some inert media or some suitable growth agar media. Enumeration of fungal spores trapped on inert media is performed by direct microscopic examination without culturing. This is referred to as total fungal spore count or non-viable analyses. When air is impacted on growth agar media any fungal spores or hyphal fragments that can germinate on that media develops into colonies (often referred to as colony forming units) that are then enumerated and identified if necessary.

Fungal Spores Trapping for Total fungal Spore Counts (Non-viable).

The common air samplers for total fungal spores counts are slit and circular type of samplers. Slit samplers include Air-O-Cell, BioCell, VersaTrap, Allergenco, BioSis, and Burkard. Examples of circular samplers include Cyclex, Cyclex-d and Micro-5. These samplers (cassettes) are attached to a pump that can draw air at the rate recommended by the manufacturer of the cassette.

Fungal spores Enumeration Using Air-O-Cell Cassettes

The Air-O-Cell cassettes are opened in the laboratory by cutting around the sealing band. The glass cover slip (containing the sample trace) is removed and slowly placed at an angle with the media collection side facing either upwards or downwards. If the glass cover slip is to be placed with the media facing down, one to two (1-2) drops of lacto phenol cotton blue is placed in the center of the microscope slide before placing the glass cover slip. The edges of the cover glass can then be secured with a drop of nail polish. If the cover glass is to be placed with the media collection side facing up, it can first be secured with nail polish and then 1-2 drops of lactophenol cotton blue is placed in the middle of the sample. A second clean cover glass is then placed to cover the sample collection media and spread out the stain. Care should be taken not to trap air bubbles as these could interfere with the analyses.

The sample deposition trace is identified at lower magnification (10X or 20X) objective and spore counting is performed at a minimum magnification of X400. There has been a lot of debate as to what magnification should be used during identification and enumeration. Some analysts have recommended use of 100X oil immersion. However, it’s important to note that use of 100X objective with oil can be very messy and in most cases may not add quality to the results. In our opinion, the most important considerations when it comes to spore enumeration and identification are a good quality microscope and a well trained analyst.

In the past laboratories were using a variety of methods to analyze spore traps. This made comparison of results (and even terms used in the reports) difficult. The ASTM International has now released a new standard “Standard Test Method for Categorization and Quantification of Airborne Fungal Structures in an Inertial Impaction Sample by Optical Microscopy”. While this standard may not resolve all limitations of spore traps it will ensure that labs that use the standard analyze samples in a similar manner. The standard, however, may not improve the precision or accuracy of results, since, as it’s stated in the standard itself “…the detector in this method is the analyst, and therefore results are subjective, depending on the experience, training, qualification, and mental and optical fatigue of the analyst”.

Enumeration and Identification of Fungal Colony Forming Units.

Enumeration of colonies is performed under a stereo microscope or a suitable colony counter. Counting of colony forming units (CFU) is straight forward and easier than enumeration of total fungal spore counts. However, accuracy may be affected by:

Colony density: If there are too many colonies, they tend to merge and this makes counting very difficult and inaccurate.

Counting is easier in some media such as dichloran 18% glycerol (DG18) agar which restricts the growth of colonies and significantly reduces the number of merging colonies. One drawback in using DG18 is that it can completely inhibit the growth of some fungi especially the hydrophilic species such as Stachybotrys and Chaetomium.

Malt extract agar (MEA) is commonly used in indoor air quality surveys. MEA is a nutrient rich medium and has high water activity, which favors hydrophilic and fast growing species which may mask, inhibit or entirely suppress the growth of other fungi.

Simultaneous use of MEA and DG18 is recommended since MEA supports the growth of a wide range of hydrophilic and mesophilic fungi while DG18 supports the growth of xerophilic and moderately xerophilic fungi. Therefore, use of MEA or DG18 alone may significantly reduce the number of species recovered.

For air samples collected using Andersen and other similar samplers, the colony count is adjusted (positive hole correction) before calculating the number of CFU per cubic meter of air. This adjustment is however not performed for air samples collected using the Reuter Centrifugal Sampler (RCS).

Non-viable Air Sampling

Indoor air sampling for airborne fungi is frequently conducted to assess the levels of fungal contamination and subsequently the potential risk to building occupants. It is also used to determine if there was hidden mould growth in the building or to determine the effectiveness of remediation procedures. One of the most cited advantages of non-viable air sampling is that detection of fungal structures (spores, hyphal fragments, etc) is not dependent on their viability or the suitability of agar media. Non-viable air samples are collected with samplers such as Air-O-Cell, Allergenco, VersaTrap, Burkard, Cyclex, Cyclex-d and Micro-5 among others. The spores (whether viable or dead) and other particulates are trapped on the sticky surface of the spore trap and can then be directly enumerated and identified under a microscope. Since both viable and nonviable spores can be enumerated, an efficient non-viable air sampler is expected to give a better estimate of the level of airborne fungal contamination than a viable air sampler.

Viable Air Sampling

Viable air samples are often collected on agar media either in strips (if using Reuter Centrifugal Sampler) or in Petri-dishes for Andersen sampler. Unlike non-viable air sampling, detection and subsequent enumeration and identification of airborne fungal particulates collected on growth media depends on whether the spores and hyphal fragments are viable and whether the media used can support their growth into colonies. For this reason, colony counts are usually lower than spore counts. Even if all the fungal structures were viable, colony counts are likely to be lower than the spore/hyphal fragment counts because what is counted as a single colony could have developed from more than a single spore or hyphal fragment. In one study it was found that the ratios between the total fungal spores collected by the Burkard sampler and the viable fungi collected by the Andersen sampler ranged between 0.29 and 7.61.

Non-viable Air Sample

Is Non-viable Fungal Air Sampling Alone Adequate? In most cases viable air sampling is only used in situations where identification of the moulds to species level is required. However, our observation in the lab seems to suggest use of spore traps alone may not be adequate for airborne fungal sampling. On many occasions we have recovered moulds in viable samples that were not observed in non-viable samples even when viable and non-viable samples were taken side by side. For example Chaetomium and Stachybotrys spores, which are fairly easy to identify from spore traps have appeared in viable samples, yet, they were not detected from the non-viable samples. We have also observed that although non-viable sampling gives higher counts than viable sampling in most cases, this is not always the case. There are many factors that can contribute to these “unexpected” results.

Conclusion

Since both non-viable and viable air sampling have limitations, using either method singly is not adequate. To obtain conclusive information on the level of contamination and the diversity of airborne fungi in a building, taking both viable and non-viable air samples is preferable. We recommend the Calgary Health Region’s protocol, “Fungal Air Testing, Investigation and Reporting Requirements for Residential Marihuana Grow Operations (Revised May 2006)”. With few exceptions, the protocol requires that fungal air sampling consist of both viable samples (e.g. RCS or similar) and non-viable samples (e.g., Air-O-Cell) taken side by side.

Mold Sampling

The mold sampling method one chooses should be determined by the objective of the investigation. One may sample air or surfaces for mold contamination. The standard method for sampling air is to use a volumetric sampler e.g. RCS or Andersen N6 for viable airborne spores and hyphal fragments and Air-O-Cell, VersaTrap, Allergenco and others such cassettes for total spore counts. Testing of surfaces may involve use of RODAC agar plates for smooth surfaces, and swabs and adhesive tape on all other surfaces. It is important to note that adhesive tapes may not work well on wet and porous surfaces. Bulk samples can also be taken and plated onto agar plates or analysed by direct microscopic examination. Dust samples can be collect from surfaces such as carpets, upholstered furniture and textiles.

Media For Mold Sampling

It’s important to select media for mold sampling wisely. If one decides to collect viable air samples, the choice of media to use is very
important. Generally, malt extract agar (MEA) is used. It is a “broad spectrum” medium that supports the growth of a wide range of fungal species. However, antibiotics may have to be incorporated to surpress bacteria growth. Its main disadvantage is that fast growing molds tend to overgrow slow growers making it difficult to count colonies. To overcome this problem, DG18 and Rose Bengal can be used. These media have compounds added to them to slow down fast growing fungi and inhibit bacterial growth. If one is sampling a relatively dry environment, MEA+40% sucrose would be recommended for detecting xerophilic (dry loving) fungi.

Mold Identification

Currently, the only reliable means for routine identification of mold species is to perform traditional mycological methods. This requires years of training and practice. Be sure to use a lab that has a qualified Mycologist on-board (preferably at PhD level). The lab should also be regularly participating in a recognised proficiency testing program such as the AIHA EMPAT program.

Performing Effective Mold Sampling

If you need to take mold samples, use properly trained personnel or to get yourself trained. If you decide to undergo training, select a mold training course that provides skills and background information to enable you recognize indoor mold, develop effective mold sampling strategies, and interpret laboratory results.

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